Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Hyper-production of l-trytophan via fermentation with crystallization

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid. Correspondence to: H. Tsunekawa     

Biosynthesis of indole-3-acetic acid in tomato shoots: Measurement,mass-spectral identification and incorporation of −2H from −2H2O into indole-3-acetic acid,d- and l-tryptophan,indole-3-pyruvate and tryptamine

Indole-3-acetic acid (IAA) and its putative precursors, l- and d-tryptophan, indole-3-pyruvate, and tryptamine were isolated from tomato (Lycopersicon esculentum (L.) Mill.) shoots, identified by mass spectrometry, and measured using capillary gas chromatography with an electron capture detector and radioactive internal standards. Average amounts present were 7.9ng · (g FW)^–-1 IAA, 5.7ng · (g FW)^–-1 indole-3-pyruvate, 132 ng · (g FW)^–-1 tryptamine, 103 ng · (g FW)^–-1 d-tryptophan, and 2250 ng · (g FW)^–-1 l-tryptophan. Indole-3-acetaldoxime was not found; detection limits were less than 1ng · (g FW)^–-1. When tomato shoots were incubated for 6, 10 and 21 h in 30% ^–2H₂O, up to four positions in IAA, l- and d-tryptophan, tryptamine and indole-3-pyruvate became labelled with ^–2H. Compounds became labelled rapidly with 10% of IAA molecules containing ^–2H after 6 h. The percentage of labelled molecules of IAA and l-tryptophan increased up to 10 h but then decreased again, correlating with an increase in the total shoot tryptophan and presumably a result of protein hydrolysis in the excised, slowly senescing tissue. The amount of ^–2H in d-tryptophan also showed an increase followed by a decrease, but the proportion of labelled molecules was much less than in l-tryptophan and IAA. Tryptamine became labelled initially at a similar rate to IAA but continued to accumulate ^–2H up to 21 h. We conclude that tryptamine is synthesized from a different pool of tryptophan from that used in IAA synthesis, and is not a major endogenous precursor of IAA in tomato shoots. Indole-3-pyruvate was the most heavily labelled compound after 6 and 10 h incubation (21-h data not available). Furthermore, the proportion of ^–2H-labelled indole-3-pyruvate molecules was quantitatively consistent with the amount of label in IAA. On the other hand, a quantitative comparison of the IAA turnover rate and the rate of ^–2H incorporation into both l- and d-tryptophan indicates that IAA is not made from the total shoot pool of either l- or d-tryptophan. Instead IAA appears to be synthesized from a restricted pool which is turning over rapidly and which has access to both newly synthesized tryptophan and that from protein hydrolysis.Abbreviations GC-ecd gas chromatography with electroncapture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - IAOX indole-3-acetaldoxime - IPyA indole-3-pyruvate - PFB pentafluorobenzyl - R_T retention time - TNH₂ tryptamine - Trp tryptophan - SIM selected ion monitoring We wish to thank Ms. Sue Alford for running the mass spectra and Dr Harry Young for advice with the mass spectrometry. The work was supported by grants from the University of Auckland Research Committee and the C. Alma Baker Trust fund. The mass spectrometer was supported jointly by the University Grants Commitee (NZ) and the DSIR Division of Horticulture and Processing.     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000     

Metabolic engineering and flux analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for L-serine production

l-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of l-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of l-serine to pyruvate and glycine, releasing the feedback inhibition by l-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH^r). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in l-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular l-serine pool reached (14.22±1.41) μmol g_CDM ⁻¹, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH^r improved the l-serine biosynthesis pathway. In addition, the flux from l-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that l-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C₁ units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for l-serine production in C. glutamicum.     

Redistribution of hepatocyte chloride during l-alanine uptake

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V _m), intracellular K⁺, Cl^-, and Na⁺ activities (a ⁱ _k, a _Cl ⁱ and a _Na ⁱ ), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA⁺) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA⁺ activity measured with the K⁺ electrode. Hepatocyte control V _m was -33±1 mV. l-alanine uptake first depolarized V _m by 2±0.2 mV and then hyperpolarized V _m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a _Na ⁱ increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a _K ⁱ did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V _m, but now a _K ⁱ decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V _m by l-alanine uptake also resulted in a 38% decrease of a _Cl ⁱ from 20±2 to 12±3 mm (P < 0.001). Changes in V _m and V _Cl — V _m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl^– with the V _m in hepatocytes. Added Ba²⁺ abolished the l-alanineinduced hyperpolarization, and a _Cl ⁱ remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a _K ⁱ is regulated by increased Na⁺-K⁺ pump activity during l-alanine uptake in spite of cell swelling and increased V _m due to increased K⁺ conductance. The hyperpolarization of V _m during l-alanine uptake provides electromotive force to decrease a _Cl ⁱ . The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Equilibrium and dynamic investigations of organic acids adsorption onto ion-exchange resins

The aim of the study was to determine properties of selected ion-exchange resins for citric and lactic acids recovery, to define sorption isotherms for these acids at different temperatures (in the range of 20–60°C) and to determine diffusion coefficients inside sorbent particles. A mathematical model of the ion-exchange process in the chromatographic column and its experimental verification is also presented. During investigations 18 types of ion-exchange resins were tested. It was found that weakly basic resins were more suitable for the recovery process than strongly basic ones. The best resin for the separation of citric acid was Amberlite IRA-67 and for lactic acid Amberlite IRA-92. As a result of transient-state sorption experiments diffusion coefficients of the citric acid inside the sorbent particle at different temperatures were obtained. It was found that D_p increased with the temperature by two times in the range of 20–60°C, and its value at 60°C was 7.2×10^–10 m²/s. The proposed mathematical model was applied to identify bed operation parameters in the column for the needs of the simulated moving bed chromatography method.List of symbols b Equilibrium constant in Langmuir equation, [dm³/g] - c Acid concentration in the liquid phase inside the particle pores, [g/dm³] - C Acid concentration in the liquid, [g/dm³] - D_L Axial dispersion coefficient, [m²/s] - D_p Intraparticle diffusion coefficient, [m²/s] - k_f Liquid film mass transfer coefficient, [m/s] - L Ion-exchanger bed height, [m] - q Acid concentration in the adsorbent phase, [g/dm³] - R_p Particle radius, [m] - U Volumetric flow rate of the feeding solution, [dm³/s] - V Volume of the solution, [dm³] - W Weight of the wet resin particles, [g] - The ion-exchanger bed porosity, [-] - _p Particle porosity, [-] - Linear liquid velocity, [m/s] - Apparent density of the wet resin, [g/dm³]     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

Production of acetone and butanol by Clostridium acetobutylicum in continuous culture with cell recycle

Summary To increase the solvent productivity of the acetone-butanol fermentation, a continuous culture of Clostridium acetobytylicum with cell recycling was used. At a dry cell mass concentration of 8 g l^-1 and a dilution rate of D=0.64 h^-1, a solvent productivity of 5.4 g l^-1 h^-1 was attained. To prevent degeneration of the culture, which occurs with high concentrations of solvents (acetone, butanol and ethanol), different reactor cascades were used. A two-stage cascade with cell recycling and turbidostatic cell concentration control turned out to be the best solution, the first stage of which was kept at relatively low cell and product concentrations. A solvent productivity of 3 and 2.3 g l^-1 h^-1, respectively, was achieved at solvent concentrations of 12 and 15 g l^-1.Symbols D Dilution rate (h^-1) - r _p solvent productivity (g l^-1 h^-1) - s residual glucose concentration (g l^-1) - V _R reactor volume (l) - V _O overall volume (l) - x (dry) cell mass concentration (g l^-1) - Y _P/S solvent yield (g g^-1)     

Amino acid transport by<Emphasis Type="Italic"> Sphingomonas</Emphasis> sp. strain Ant 17 isolated from oil-contaminated Antarctic soil

The Antarctic bacterial isolate Sphingomonas sp. strain Ant 17 utilized a wide range of L-isomer amino acids as the sole carbon and energy source for growth. The pH and temperature optima for growth on amino acids were pH 7.0 and 15°C, respectively. Growth on serine and tryptophan was inhibited by uncouplers and inhibitors of oxidative phosphorylation, but not by monensin, a Na⁺/H⁺ antiporter, suggesting that sodium gradients were not specifically required for growth on these amino acids. Serine transport was via a high-affinity (apparent K_m of 8 M) permease specific for both the L- and D-isomer. Tryptophan transport exhibited biphasic kinetics with both high-affinity (apparent K_m of 2.5 M) and low-affinity (non-saturable) uptake systems detected. The high-affinity system was specific for L-tryptophan, L-tyrosine, and L-phenylalanine whereas the low-affinity permease was specific for L-tryptophan and L-phenylalanine, but not L-tyrosine. Neither orthovanadate nor sodium arsenate, inhibitors of ATP-dependent permeases, had any significant inhibitory effect on the rate of serine and tryptophan transport. The protonophore carbonyl cyanide m-chlorophenylhydrazone completely abolished serine and tryptophan transport; maximum rates of solute uptake were observed at acidic pH values (pH 4.0–5.0) for both amino acids. These results suggest that an electrochemical potential of protons is the driving force for serine and tryptophan transport by Ant 17. These high-affinity proton-driven permeases function over environmental extremes (e.g. broad temperature and pH range) that are likely to prevail in the natural habitat of this bacterium.     

Amino acid transport system y+L of human erythrocytes: Specificity and cation dependence of the translocation step

The transport specificity of system y⁺L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of l-[¹⁴C]-lysine. Some neutral amino acids, l-lysine and l-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external l-leucine and l-lysine increased the rate by 5.3±0.63 and 6.2±0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (l-tryptophan, l-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (l-alanine, l-serine). Half-saturation constants (±sem) calculated from this effect (l-lysine, 10.32±0.49 m and l-leucine, 11.50±0.50 m) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na⁺, Li⁺ or K⁺ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na⁺ or Li⁺ were replaced by K⁺. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.We wish to thank Dr C.A.R. Boyd for helpful discussions and Prof. H.N. Christensen for sharing with us very relevant bibliographic material. We are grateful to FONDECYT (1282/91) and DTI (B 2674) (Chile) for financial assistance.     

Kinetic determination of tryptophan by using the B-Z oscillating chemical system

A simple and rapid method was devised for determination of tryptophan, based on the Belousov-Zhabotinskii (B-Z) oscillating chemical system. Changes in oscillating period and amplitude were linearly proportional to the negative logarithm of l-tryptophan concentration over the range of 6.44 × 10⁻⁷–2.55 × 10⁻⁴ M, with the regression coefficients of near unity and a lower detection limit of 6.5 × 10⁻⁸ M. d-tryptophan was also examined although it is rarely found in most biological fluids, and perhaps not at all in natural proteins. The change of period against to negative logarithm of d-tryptophan concentration over the range of 4.9 × 10⁻⁵–8.24 × 10⁻⁴ M is linear. Because the optimum conditions for determination of l- and d-tryptophan are not the same, a little amount of d-tryptophan does not affect the determination of l-tryptophan. Various influences were studied and a possible mechanism of perturbation to the B-Z oscillator by tryptophan was also discussed. Spectrophotometry and fluorescence spectrophotofluorimetry were used for comparision and confirmation of the results.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

Tryptophan Synthase and Production of l-Tryptophan in Regulatory Mutants

A 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, No. 187, accumulated 2.6 g of indole 3-glycerol (InG) in addition to 8.0 g of l-tryptophan per liter in the culture medium. The addition of l-serine to the medium decreased the accumulation of InG and increased that of l-tryptophan up to a concentration of 10.3 g/liter, while the addition of l-tryptophan increased the InG accumulation, suggesting that InG was formed by hydrolysis of indole 3-glycerol phosphate (InGP), the substrate of tryptophan synthase (TS) which catalyzed the final step reaction of tryptophan biosynthesis. Then, in order to examine the mechanism of the InG accumulation, TS was purified from tryptophan auxotroph, TA-60. The reaction mechanism of TS was Ordered Bi Bi with Km’s of 0.63 and 0.038 mm for serine and InGP, respectively. Tryptophan, a product of the TS reaction, inhibited TS competitively with respect to serine and the Ki for tryptophan was estimated to be 2.0 mm. On the other hand, anthranilate synthase (AS), the first enzyme in the tryptophan biosynthetic pathway, was much less sensitive to the feedback inhibition by tryptophan in strain No. 187 than in the wild strain. The tryptophan concentration giving 50% inhibition of AS in strain No. 187 was estimated to be 2.4 mm, almost comparable to that of TS, 7.7 mm. From these results, it was concluded that the accumulation of InG in strain No. 187 would result from the product inhibition of TS by the tryptophan accumulated.     

Spectroscopic,theoretical and structural characterization of hydrogensquarates of <Emphasis Type="SmallCaps">l</Emphasis>-threonyl-<Emphasis Type="SmallCaps">l</Emphasis>-serine and <Emphasis Type="SmallCaps">l</Emphasis>-serine

Summary. Hydrogensquarates of dipeptide l-threonyl-l-serine (H-Thr-Ser-OH) and l-serine (HSq × Ser) have been synthesized, isolated and spectroscopic characterized by solid-state linear-polarized IR-spectroscopy, ¹H- and ¹³C-NMR, ESI-MS and HPLC with tandem masspectrometry (MS-MS) methods. The structures of the salts and neutral dipeptide have been predicted theoretically by ab initio calculations. In the case of H-Thr-Ser-OH the theoretical data are supported by IR-LD ones. The hydrogensquarates consist in positive charged dipeptide or amino acid moiety and negative hydrogensquarate anion (HSq) stabilizing by strong intermolecular hydrogen bonds. The data about the l-serine hydrogensquarate are compared with known crystallographic data thus indicating a good correlation between the theoretical predicted structures and experimentally obtained by single crystal X-ray diffraction.     

Regulation of the synthesis of enzymes of tryptophan dissimilation in Acinetobacter calcoaceticus

Summary Acinetobacter calcoaceticus dissimilates tryptophan via the -ketoadipate pathway. The first enzyme, tryptophan oxygenase (l-tryptophan: oxygen oxidoreductase; EC 1.13.1.12), is substrate-induced by tryptophan. The second two enzymes, formamidase (aryl-formylamine amidohydrolase; EC 3.5.1.9) and kynureninase (l-kynurenine hydrolase; EC 3.7.1.3), are induced by the next intermediate, kynurenine. The last enzyme specific to tryptophan dissimilation, anthranilate oxidase, is substrate induced. This inductive pattern is in marked contrast to the extensive coordinacy of enzyme synthesis characteristic of the remainder of the -ketoadipate pathway.     

Whole body autoradiographic study on the distribution of14C-d-serine administered intravenously to rats

Summary The distribution of radioactivities in rats following intravenous administration of¹⁴C-d- or -l-serine was investigated by whole body autoradiography. The radioactivities were distributed throughout the whole body in both cases with the greatest amount being found in the pancreas. D- andl- Serine levels in the pancreas were determined by high-performance liquid chromatography with a chiral column which revealed, for the first time, the existence ofd-serine in the rat pancreas (12.6 ± 7.90 nmol/g wet tissue) together with a much higher concentration (924 ± 116 nmol/g) ofl-serine. The results suggested that exogenous D-serine of dietary origin contributed at least in part to the D-serine levels found in mammalian tissues.The accumulation of radioactivity in the kidney, especially in the corticomedullary area, even at 24 hr after administration of¹⁴C-l-serine suggested a possible link between acute necrosis of the renal proximal tubules and the administration of a large dose of D-serine [Am J Pathol 77: 269–282 (1974)].     

Procedure for determination of elemental sulphur bio-oxidation rate

A procedure is described for measuring the rate of biooxidation of elemental sulphur in nutrient solutions. Results of preliminary measurements of sulphur bio-oxidation rate in a dynamic system are presented. The rate of sulphur bio-oxidation has been determined at the level of 0.02–0.05 g of sulphur per m² of sulphur per h.List of Symbols C g/dm³ concentration of sulphate ions - C ₂ g/dm³ concentration of sulphate ions in withdrawn solution - C g/dm³ C difference between solution outlet and inlet to sulphur bed - F m² sulphur surface exposed to bacteria action - m g mass of elemental sulphur - V dm³ volume of solution - V ₀ dm³/h volume of fresh solution supplied to the set - V ₁ dm³/h circulating solution flow rate - V ₂ dm³/h volume of solution withdrawn - h time Abbreviations RBES rate of bio-oxidation of elemental sulphur     

Studies on RTD and continuous culture (SCP) in cylindrical and tapered reactors

The performance of a tapered reactor for the continuous cultivation of bakers' yeast (SCP) from cane molasses has been compared with that of a conventional cylindrical reactor. It is found that the tapered reactor has less non-idealities (bypass and deadspace).Using the experimentally evaluated bypass and deadspace values, a model for predicting conversions of substrate (cane molasses), based on the RTD model proposed by Cholette and Cloutier has been developed. The experimental substrate conversions are found to match the model satisfactorily.List of Symbols D h^–1 dilution rate - E() exit age distribution function - K _s kg/m³ Monod's saturation constant - -r _sa kg/(m³ · h) rate of substrate utilization - S kg/m³ substrate concentration expressed as dextrose equivalent (DE) - S _a kg/m³ substrate concentration in active zone - S ₀ kg/m³ initial substrate concentration - S/S ₀ dimensionless substrate concentration - v _a dm³/h volumetric flow through active zone - v _b dm³/h volumetric flow through bypass stream - u _l dm³/h substrate feed rate - v _g dm³/min air-flow rate - V dm³ total working volume of the reactor - V _a dm³ volume of active zone in reactor - V _d dm³ volume of dead zone in reactor - X kg/m³ biomass concentration Greek Letters fraction of bypass of feed, v _b /v _l - fraction of deadspace, V _d /V - dimensionless residence time - _m h^–1 maximum specific growth rate - h mean residence time, V/v _l